Pachence J M, Blasie J K
Department of Chemistry, University of Pennsylvania, Philadelphia 19104.
Biophys J. 1987 Nov;52(5):735-47. doi: 10.1016/S0006-3495(87)83268-X.
X-ray diffraction and spectroscopic techniques were used to characterize ultrathin fatty acid multilayers having a bound surface layer of cytochrome c. Three to six monolayers of arachidic acid were deposited onto an alkylated glass surface, using the Langmuir-Blodgett method. These fatty acid multilayer films were stored either in a 1 mM NaHCO3 pH 7.5 solution or a buffered 10 microM cytochrome c solution, pH 7.5. After washing extensively with buffer, these multilayer films were assayed for bound cytochrome c by optical spectroscopy. It was found that the cytochrome c bound only to the odd-numbered monolayer films (which have hydrophilic surfaces). The theoretical number of cytochrome c molecules bound to the ultrathin multilayer films having three or five monolayers was calculated as N = 1.2 x 10(13)/cm2 (assuming a hexagonally close-packed monolayer of protein), which would produce an optical density of 0.002 at a wavelength of 550 nm; for a three or five monolayer ultrathin film that was incubated with cytochrome c, OD550 approximately equal to 0.002. The protein was released from the film when treated with greater than 100 mM KCl solution, as would be expected for an electrostatic interaction. Meridional x-ray diffraction data were collected from the arachidic acid films with and without a bound cytochrome c layer. A box refinement technique, previously shown to be effective in deriving the profile structures of nonperiodic ultrathin films, was used to determine the multilayer electron density profiles. The electron density profiles and their autocorrelation functions showed that bound cytochrome c resulted in an additional electron dense feature on the multilayer surface, consistent with a bound cytochrome c monolayer. The position of the bound protein relative to the multilayer surface was independent of the number of fatty acid monolayers in the multilayer. Future studies will use these methods to investigate the structures of membrane protein complexes bound directly to the surface of multilayer films.
采用X射线衍射和光谱技术对具有细胞色素c结合表面层的超薄脂肪酸多层膜进行表征。使用朗缪尔-布洛杰特方法将三到六个花生酸单分子层沉积在烷基化玻璃表面上。这些脂肪酸多层膜分别储存在pH 7.5的1 mM碳酸氢钠溶液或pH 7.5的10 μM细胞色素c缓冲溶液中。用缓冲液充分洗涤后，通过光谱法测定这些多层膜中结合的细胞色素c。发现细胞色素c仅与奇数单分子层膜（具有亲水性表面）结合。计算出结合到具有三个或五个单分子层的超薄多层膜上的细胞色素c分子的理论数量为N = 1.2×10¹³/cm²（假设蛋白质为六方密堆积单分子层），这将在550 nm波长处产生0.002的光密度；对于与细胞色素c孵育的三个或五个单分子层的超薄膜，OD550约等于0.002。如预期的静电相互作用那样，当用大于100 mM的氯化钾溶液处理时，蛋白质从膜中释放出来。收集了有无结合细胞色素c层的花生酸膜的子午线X射线衍射数据。一种先前已证明在推导非周期性超薄膜的轮廓结构方面有效的盒式细化技术被用于确定多层电子密度轮廓。电子密度轮廓及其自相关函数表明，结合的细胞色素c在多层表面上产生了一个额外的电子致密特征，这与结合的细胞色素c单分子层一致。结合蛋白相对于多层表面的位置与多层中脂肪酸单分子层的数量无关。未来的研究将使用这些方法来研究直接结合到多层膜表面的膜蛋白复合物的结构。
